Traduccion Articulos VCD Thomas R. Rizzo

8/2/2019 Traduccion Articulos VCD Thomas R. Rizzo

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Spectroscopic studies of cold, gas-phase biomolecular ionsThomas R. Rizzo*, Jaime A. Stearns and Oleg V. BoyarkinLaboratoire de Chimie Physique Moleculaire, Ecole Polytechnique Federale de Lausanne,CH-1015 Lausanne, Switzerland(Received 13 April 2009; final version received 20 May 2009)While the marriage of mass spectrometry and laser spectroscopy is not new,developments over the last few years in this relationship have opened up newhorizons for the spectroscopic study of biological molecules. The combinationof electrospray ionisation for producing large biological molecules in the gasphase together with cooled ion traps and multiple-resonance laser schemes areallowing spectroscopic investigation of individual conformations of peptideswith more than a dozen amino acids. Highly resolved infrared spectra of singleconformations of such species provide important benchmarks for testing theaccuracy of theoretical calculations. This review presents a number oftechniques employed in our laboratory and in others for measuring thespectroscopy of cold, gas-phase protonated peptides. We show examples thatdemonstrate the power of these techniques and evaluate their extension to stilllarger biological molecules.Keywords: gas-phase biological molecules; cooled ion traps; electrospray;infrared spectroscopyContents page1. Introduction 4822. Experimental and computational approach 4842.1 Overview 4842.2 Ion generation 4852.3 Cooling and trapping 4852.4 Spectroscopic interrogation 4862.5 Computational methods 4873. Spectroscopy of protonated aromatic amino acids 4884. The scaling of spectral complexity with increasing molecular size 4945. Spectroscopy of helical peptides 4956. Toward still larger molecules 503*Corresponding author. Email: [email protected] 0144 235X print/ISSN 1366 591X online

_ 2009 Taylor & FrancisDOI: 10.1080/01442350903069931http://www.informaworld.comDownloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

7. Limitations and future directions 5078. State of comparison with theory 5099. Conclusions 511Acknowledgements 512References 5121. IntroductionBecause the functions of biological molecules are intimately connected to theirthreedimensionalstructure, an enormous amount of effort is expended to determine the latterwith the hope of understanding, and perhaps intervening in, the former. While

biomolecular structure is determined primarily by x-ray crystallography and NMR,theory is playing an increasingly important role. As available computational powercontinues its exponential rise, increasingly large molecules become amenable to theoreticaltreatment. In addition to its potential predictive power, an accurate theoretical descriptionof biological molecules allows one to dissect and analyse the counterbalancing forces thatcontrol their structure as well as to examine the properties of individual stable conformers.To make sure that theoretical treatments properly describe the underlying physics and arecapable of reliably predicting structures, comparison with benchmark experiments isessential. Perhaps the most rigorous test is to compare theory and experiment on isolatedbiological molecules, free from the complicating effects of solvent.In the mid-1980s molecular spectroscopists began to remove biological molecules fromtheir natural environment to interrogate them in the cold, isolated environment ofsupersonic molecular beams [1 9]. Early experiments used electronic spectroscopy to

investigate the amino acid tryptophan with the goal of determining the number of stableconformations that it could adopt in vacuo [1 4]. This was driven by observations that thefluorescence decay rate of tryptophan in solution, which is used as a monitor of protein


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folding, is non-exponential [10 12], and one of the models to explain these observationspostulated the existence of different conformers having different fluorescence quenching rates[12]. Electronic spectra of isolated tryptophan did indeed identify a number of stableconformations[4], although at the time theoretical methods were not able to makereasonable predictions for these structures. Today the conformations of isolated aminoacids can be easily and reliably calculated.

The earliest experiments on isolated amino acids [1 4] and their derivatives [8,13,14]used simple heating to put them in the gas phase. With the advent of laser desorptiontechniques, larger, more complex molecules could be volatilised without decomposition[5 7, 15], and this helped ignite an explosion in spectroscopic studies of neutral peptideswith up to 15 amino acid residues [5,6,9, 16 29]. The increasing complexity of thesemolecules require more sophisticated spectroscopic techniques to extract structuralinformation, and this has been achieved by employing IR UV [18 21,23,24,26, 30 40],UV UV [30,33,36,41] and even IR IR UV multiple-resonance methods [42] together withcooling in supersonic expansions.482 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

Still larger biological molecules become increasingly difficult to produce in the gasphase via thermal techniques without decomposition. The advent of MALDI [43,44] andelectrospray [45,46] in mass spectrometry has allowed intact biological molecules ofvirtually any size to be put into the gas phase in the form of closed-shell molecular ions andopened up new avenues of investigation. Bowers and co-workers used these techniquestogether with ion mobility to characterise the average cross section of such species [47,48].Wang and co-workers measured photoelectron spectra of multiply-charged, non-biologicalanions generated by electrospray [49], and Parks and co-workers observed fluorescencefrom cytochrome C in an electrospray plume [50] and later from small proteins in aquadrupole ion trap [51]. However it was Andersen et al. [52,53] who first used absorption/ action spectra to characterise closed-shell biomolecular ions produced by electrospray.These groundbreaking experiments measured electronic absorption spectra of greenfluorescent protein chromophore in an electrostatic ion storage ring by detecting theneutral products subsequent to either photodetachment (in the case of anions) orphotofragmentation (in the case of cations). The spectra they obtained exhibited onlybroad structure, although this is likely due to the warm internal temperatures of the ions inthe storage ring. At about the same time, McLafferty et al. [54,55] demonstrated that onecould do infrared multiphoton dissociation (IRMPD) of electrosprayed peptides andproteins in a Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometerusing an optical parametric oscillator (OPO) operating in the light-atom stretch region.Tuning the frequency of their IR OPO produced photofragment excitation spectra thatreflect the infrared absorption of OH and NH stretch vibrations. Even though the ions intheir trap were at room temperature, the observed bands were about 30 cm _1 wide, whichis sufficiently narrow to distinguish many light atom stretch bands. Von Helden et al. [56]extended IRMPD spectroscopy into the amide I and II regions of the infrared using a freeelectron laser to measure infrared spectra of potassiated cytochrome C ions formed byelectrospray, where detection of infrared absorption was monitored by the loss ofthe potassium adduct. Although the individual bands in these spectra were rather broad

(30 50 cm _1

), the overall vibrational pattern in this region suggests a large degree of _-helical content. Shortly thereafter, Kamariotis et al. [57] measured infrared spectra ofsolvated amino acid ions to look for evidence of zwitterion formation by detecting solventloss subsequent to IR absorption. These spectra were used to test the claim that afteradding 3 4 water molecules a cationised amino acid exists in a salt-bridge arrangementwith the amino acid in a zwitterionic form [58].While all these studies broke new ground in providing spectroscopic data to helpcharacterise the conformational properties of closed-shell biomolecular ions, the spectralcomplexity was substantially greater and the resolution correspondingly lower than studiesof neutrals cooled in supersonic expansions. Simons et al. [59 61] developed aphotochemical method for producing protonated biological molecules in a supersonicexpansion, and while perhaps not the most widely applicable, this approach demonstratedthat the spectra of closed-shell ions are inherently no more complex than their

corresponding neutrals. As the size and spectral complexity of the molecules increase,cooling the molecules is simply necessary to facilitate spectroscopic analysis.In a major step toward spectroscopic studies of cold, biomolecular ions, Weinkauf


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et al. [62] measured the electronic excitation spectrum of protonated tryptophan producedby electrospray and stored in a liquid-nitrogen-cooled quadrupole ion trap. While theInternational Reviews in Physical Chemistry 483Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

electronic spectrum they obtained showed some structure, the spectral features were ordersof magnitude broader than molecules cooled in a supersonic expansion, leading one toquestion the cooling efficiency in their trap. It was later discovered that the spectrum ofprotonated tryptophan is inherently broad due to fast excited state dynamics [63].Nevertheless, Weinkaufs [62] experiments served as a major stimulus to study electrosprayed ions in cold traps.This present article reviews the work currently being done in the authors laboratory combining electrospray ionisation for producing gas-phase biomolecules together withcollisional cooling in a cold 22-pole ion trap and IR UV double-resonance techniques tosimplify the spectra. The primary goals of this work have been to understand the intrinsicproperties of biological molecules and provide critical benchmark tests of theory, and forthis we look at bare, isolated molecules in the gas phase. While as a test of theory, thespectroscopy of gas-phase biomolecules has relevance completely apart from theircondensed phase analogs, the closed-shell molecular ions produced by electrospray are thesame species found in solution, where most biomolecules carry a net charge. Moreover, byexamining partially solvated molecules in the gas phase, we can begin to understand therole of solvent on those properties.2. Experimental and computational approach2.1. OverviewWe perform our experiments in a home-built ion-trap mass spectrometer, shownschematically in Figure 1. Rather than give a detailed description of this apparatus,which can be found in recent publications [64 70], we provide a brief overview of theexperimental approach followed by an explanation of key aspects of the experimentaldesign.Figure 1. [Colour online] Schematic of tandem mass spectrometer with a cooled, 22-pole ion trap.Adapted with permission from [86]. Copyright 2006 American Chemical Society.484 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

We use a nanospray source to produce the protonated peptides from an acidified

water/methanol solution directly into the atmosphere, where they are drawn into vacuumby a metal-coated glass capillary. The expansion that occurs at the exit of the capillarypasses through a 1mm diameter skimmer and into a hexapole ion trap, where the ions arecollected for _50 ms. Once released from the hexapole, the resulting ion packet passesthrough a quadrupole mass filter to select those of a particular m/z. The transmitted ionsare then turned 90 _ by a static quadrupole deflector, decelerated by a series of lenses andguided by an octopole ion guide into a 22-pole ion trap, which is cooled to _6K by aclosed-cycle refrigerator. Before the ion packet arrives in the trap, we inject a pulse ofhelium, giving it sufficient time to equilibrate to the temperature of the trap housing. Theions collide with the cold helium and fall in to the effective potential well of the trap. Wegive the ions approximately 10 ms to cool and then wait an additional 30 ms to pump outthe helium before we interrogate them spectroscopically.We pass a UV laser pulse from a frequency-doubled dye laser down the axis of the

22-pole ion trap, promoting the molecules to an excited electronic state from which somefraction of them dissociate. We then release both the parent and fragment ions from thetrap, turn them 90 _ in a second static quadrupole and pass them through an analysingquadrupole to select an m/z of either a particular fragment or of the parent ions. Thetransmitted ions are then detected by a channeltron electron multiplier. An electronicspectrum is obtained by recording the ion signal of a particular fragment as a function ofthe frequency of the UV laser. As is described more fully below, we measure infraredspectra of the trapped biomolecular ions using IR UV double resonance.The trapping cycle of the ions is repeated at 20 Hz. We switch the analysing quadrupoleto transmit either the fragment or the parent on alternate trapping cycles and use theparent signal from one shot to normalise the fragment signal from the subsequent one,which removes long-term fluctuations in the nanospray source.2.2. Ion generationPutting biological molecules into the gas-phase via electrospray has many advantages forspectroscopic studies. Because they are produced near room temperature and at


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atmospheric pressure, there is essentially no fragmentation. There seems to be no limitto the size of molecules that can be produced those ranging from individual amino acidsto entire viruses [71 73] have been successfully injected into mass spectrometers byelectrospray. Because we are working with charged biomolecules as opposed to neutrals,we can select their mass and control their trajectories, allowing us to guide and/or trapthem. Electrospray generates closed-shell molecular ions, either protonated or deprotonated,

which is precisely what is found in solution. The disadvantage of studyingbiomolecular ions is the relatively low number density, which is a function both of theelectrospray process itself as well as space charge effects inside the ion trap.2.3. Cooling and trappingThe linear, 22-pole ion trap is the key element of our experimental apparatus, allowingus to store the molecules and cool them through collisions with cold helium. Gerlich et al.[74 76] pioneered the use of cold, RF ion traps and applied them principally to study theInternational Reviews in Physical Chemistry 485Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

spectroscopy and chemical reaction kinetics of small ions of astrophysical importance.There is nothing particularly special about the choice of 22 poles it is simply important tohave a sufficiently large number of them so that the effective radial potential is flat for asubstantial fraction of the trap diameter [76]. As the ions ride up the radial potential wallwhen they approach the poles, they undergo oscillatory motion driven by the RF field, andcollisions with helium at this point could warm the ions as opposed to cooling them. Thusa flat radial potential with steeper walls should facilitate collisional cooling of ions, since inthis case the oscillatory motion and hence warming collisions, will be limited to a smallfraction of the trap volume. The drawbacks to using a larger number of poles is that theions spread over a larger volume resulting in a lower ion density on the axis of the trap,and optical access to the trapped ions in the radial direction is limited.2.4. Spectroscopic interrogationWe typically inject 10,000 ions into the 22-pole trap, which has an active volume of _5 cm.Given the ion density of _2_10 3 cm _3 , it is clear that any kind of direct absorptionspectroscopy would be difficult if not impossible. Moreover, this ion density is at the limitof what can be detected by laser-induced fluorescence (LIF), a problem that iscompounded by the unfavourable geometry of the 22-pole ion trap for photon detection.We thus need a more sensitive means of spectroscopic detection, and for this we usephotofragmentation.After UV excitation of an aromatic chromophore in a protonated peptide, somefraction of the ions will fragment, since quantum yields for fluorescence are rarely unity.This can occur either following internal conversion to highly excited vibrational levels ofthe ground electronic state or by crossing to other electronic states that are dissociative insome coordinate. We thus use mass-resolved detection of photofragments as a sensitivespectroscopic tool. As shown schematically in Figure 2a, we measure electronic spectra ofthe cold ions in the trap by detecting a particular charged photofragment as a function ofthe UV wavelength.Our ultimate goal is to measure infrared spectra of biomolecular ions, since suchspectra can provide information on the conformation of the molecule. Using an IR UVdouble-resonance scheme, illustrated schematically in Figure 2b, we can measure an IR

spectrum of individual conformers of the trapped ions. To do this, we fire an infrared pulsefrom an OPO 200 ns before the UV pulse, depleting the ground state population each timethe infrared frequency coincides with a vibrational transition of the parent molecule. TheIR transition is then detected as a dip in the UV photofragmentation signal as the IRfrequency is scanned through a vibrational band. For this approach to work, the moleculespromoted to a vibrationally excited state must not absorb a UV laser photon of the samefrequency as that of the ground state and produce fragments, or if they do, it must be withreduced efficiency. Because the IR-excited molecules will be internally warm, we observetheir UV absorption spectrum is substantially broadened and the peak absorption at theUV wavelength is greatly reduced, leading to a dip in the IR spectrum.While this double-resonance approach imposes the requirement that the molecule ofinterest have a UV chromophore, it has the great advantage that the UV spectrum can beused to select a particular conformer, since different conformers often have slightly

different UV spectra that can be resolved if the molecules are internally cold. Thus, the IR486 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010


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Los estudios espectroscpicos de los iones de fro, biomolecular en fase gaseosaThomas R. Rizzo *, Jaime A. Stearns y Oleg V. BoyarkinLaboratoire de Chimie Physique Mole'culaire, E'cole Fe'de'rale Polytechnique de Lausanne,CH-1015 Lausanne, Suiza(Recibido el 13 de abril de 2009, versin final recibi el 20 mayo de 2009)

Si bien el matrimonio de la espectrometra de masas y la espectroscopia lser no es nueva,los acontecimientos de los ltimos aos en esta relacin se han abierto nuevashorizontes para el estudio espectroscpico de molculas biolgicas. La combinacinde ionizacin de electrospray para la produccin de grandes molculas biolgicas en el gasfase, junto con las trampas de iones enfriados y mltiples esquemas de resonancia lserque permita una investigacin espectroscpica de conformaciones individuales de los pptidoscon los cidos ms de una docena de aminocidos. Espectros de infrarrojo altamente resueltode una sola conformaciones de estas especies constituyen puntos de referencia importantespara las pruebas de la exactitud de los clculos tericos. Esta revisin presenta una serie detcnicas empleadas en nuestro laboratorio y en otros para medir laespectroscopia de fro, en fase gaseosa pptidos protonada. Se muestran ejemplos que

demostrar el poder de estas tcnicas y evaluar su extensin a los todavagrandes molculas biolgicas.Palabras clave: fase gaseosa las molculas biolgicas, las trampas de iones enfriados;electrospray;espectroscopia de infrarrojoContenido de la pgina1. Introduccin 4822. Enfoque experimental y computacional 4842.1 Resumen 4842.2 Ion generacin de 4852.3 Refrigeracin y captura 4852.4 espectroscpicos interrogatorios 4862.5 Mtodos computacionales 4873. Espectroscopia de protonadas aminocidos aromticos 4884. La escala de la complejidad del espectro con el aumento del tamao molecular 4945. Espectroscopia de pptidos helicoidales 4956. Hacia las molculas an mayores 503* Autor para la correspondencia. Correo electrnico: @ thomas.rizzo epfl.chISSN 0144-235X de impresin / ISSN 1366-591X en lnea _ 2009 Taylor & FrancisDOI: 10.1080/01442350903069931http://www.informaworld.comDescargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 20107. Limitaciones y las direcciones futuras 5078. Estado de comparacin con la teora 5099. Conclusiones 511Agradecimientos 512Referencias 5121. IntroduccinDado que las funciones de las molculas biolgicas estn ntimamente conectados con sus tresdimensionesestructura, una enorme cantidad de esfuerzo se realiza para determinar la ltimacon la esperanza de entender, y quizs intervenir en la primera. Mientras queestructura biomolecular est determinada principalmente por cristalografa de rayos X y RMN,La teora est desempeando un papel cada vez ms importante. Como la potencia de clculodisponible


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contina con su crecimiento exponencial, cada vez ms grandes molculas susceptibles de sertericasel tratamiento. Adems de su potencial poder de prediccin, una descripcin terica precisade las molculas biolgicas permite diseccionar y analizar las fuerzas de contrapeso queel control de su estructura, as como para examinar las propiedades de los distintos

confrmeros estables.Para asegurarse de que los tratamientos tericos adecuadamente describir la fsica subyacentey secapaz de predecir de forma fiable las estructuras, la comparacin con los experimentos dereferencia esesenciales. Quizs la prueba ms rigurosa es la comparacin de la teora y la experimentacinen aisladoslas molculas biolgicas, libre de los efectos que complica de disolvente.En el espectroscopistas mediados de 1980 comenz molecular para eliminar las molculasbiolgicas desu medio ambiente natural para interrogarlos en el ambiente fro, aislado de

supersnico haces moleculares [1-9]. Los primeros experimentos utiliz la espectroscopiaelectrnicainvestigar el aminocido triptfano, con el objetivo de determinar el nmero de estableconformaciones que puede adoptar en el vaco [1-4]. Esto fue impulsado por las observacionesque eltasa de decaimiento de fluorescencia de triptfano en la solucin, que se utiliza como unmonitor de la protenaplegado, no es exponencial [10-12], y uno de los modelos para explicar estas observacionespostul la existencia de diferentes confrmeros "con extincin de fluorescencia diferenteslas tasas [12]. Los espectros electrnicos de triptfano aislados, efectivamente, identificar unaserie de estableconformaciones [4], aunque en el momento de mtodos tericos no fueron capaces de hacerpredicciones razonables para estas estructuras. Hoy en da la conformacin de aminocidosaisladosLos cidos pueden ser fcil y confiablemente calculado.Los primeros experimentos en aminocidos aislados [1-4] y sus derivados [8,13,14]utiliza calentamiento simple para ponerlos en la fase gaseosa. Con la llegada de desorcin porlsertcnicas, molculas ms grandes y complejas pueden ser volatilizados sin descomposicin[15 5-7], y esto ayud a encender una explosin en los estudios espectroscpicos de pptidosneutralcon hasta 15 residuos de aminocidos [5,6,9, 16-29]. La creciente complejidad de estasmolculas requieren tcnicas espectroscpicas ms sofisticados para extraer estructurales, la informacin, y esto se ha logrado mediante el empleo de IR-UV [18-21,23,24,26, 30-40]UV UV-[30,33,36,41], e incluso IR-IR-UV de mltiples mtodos de resonancia [42], junto conde enfriamiento en las expansiones supersnicas.482 T. R. Rizzo et al.Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010An ms grande de molculas biolgicas cada vez ms difcil de producir en el gasfase a travs de tcnicas termales sin descomposicin. El advenimiento de MALDI [43,44] yelectrospray [45,46] en la espectrometra de masas ha permitido intactas las molculasbiolgicas deprcticamente cualquier tamao que se pondrn en la fase de gas en forma de capa cerradaiones moleculares yabri nuevas vas de investigacin. Bowers y colaboradores utilizan estas tcnicas junto con la movilidad de iones para caracterizar la seccin transversal media de estas especies


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[47,48].Wang y sus colaboradores midieron los espectros de fotoelectrones de multiplicar cargado, nobiolgicosaniones generados por electrospray [49], y Parques y compaeros de trabajo observfluorescencia

de citocromo C en un penacho de electrospray [50] y ms tarde a partir de pequeas protenasen uncuadrupolo trampa de iones [51]. Sin embargo, fue Andersen et al. [52,53], quien utiliz porprimera vez la absorcin /espectros de accin para caracterizar con cscara cerrada iones biomoleculares producido porelectrospray.Estos experimentos innovadores medir los espectros de absorcin electrnica de color verdecromforo protena fluorescente en un anillo de iones de almacenamiento electrostticomediante la deteccin de laproductos neutros o con posterioridad a photodetachment (en el caso de aniones) ophotofragmentation (en el caso de cationes). Los espectros obtenidos se exhiben slo

estructura general, aunque esto es probablemente debido a las altas temperaturas internas delos iones enel anillo de almacenamiento. Al mismo tiempo, McLafferty et al. [54,55] demostraron que unapoda hacer por infrarrojos disociacin multifotnica (IRMPD) de los pptidos y electrosprayedlas protenas de una transformada de Fourier de iones de resonancia ciclotrn (FT-ICR)espectrmetro de masasutilizando un oscilador ptico paramtrico (OPO) que operan en la regin se extienden la luz-tomo.Sintonizacin de la frecuencia de su IR OPO producido photofragment espectros de excitacinquereflejan la absorcin infrarroja de las vibraciones se extienden OH y NH. A pesar de que losiones enla trampa estaban a temperatura ambiente, las bandas se observaron alrededor de 30 cm_1de ancho, quees lo suficientemente estrecha para distinguir la luz muchas bandas elsticas tomo. VonHelden et al. [56]espectroscopia extendido IRMPD en la amida I y II regiones del infrarrojo utilizando una librelser de electrones para medir los espectros infrarrojos de potassiated citocromo C formadopor ioneselectrospray, que se monitoriz la deteccin de absorcin infrarroja por la prdida deel aducto de potasio. A pesar de las bandas individuales en esos espectros fueron bastanteamplio(30-50 cm_1), el patrn general de vibracin en la regin sugiere un alto grado de _-Helicoidal contenido. Poco despus, Kamariotis et al. [57] midieron los espectros infrarrojosdesolvatados iones de aminocidos para buscar evidencia de la formacin de zwitterionmediante la deteccin de disolventesconsecuente prdida de absorcin de infrarrojos. Estos espectros se utilizaron para probar laafirmacin de que despus dela adicin de molculas de agua 3-4 un aminocido cationised existe en un acuerdo de sal-puentecon el aminocido en forma de iones hbridos [58].Aunque todos estos estudios abri un nuevo camino en el suministro de los datosespectroscpicos para ayudar acaracterizar las propiedades conformacionales de iones biomoleculares con cscara cerrada, elespectro


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complejidad era mucho mayor y la resolucin proporcionalmente menor que en los estudiosde los neutrales se enfri en expansiones supersnicas. Simons et al. [59-61] desarrollaron unmtodo fotoqumico para la produccin de molculas biolgicas protonado en un supersnicode expansin, y aunque quiz no el ms ampliamente aplicable, este enfoque ha demostradoque los espectros de capa cerrada iones no son inherentemente ms complejos que sus

neutrales correspondientes. A medida que el tamao y la complejidad del espectro delaumento de las molculas,enfriamiento de las molculas no es ms que necesaria para facilitar el anlisisespectroscpico.En un gran paso hacia los estudios espectroscpicos de iones de fro, biomolecular, Weinkauf et al. [62] midieron el espectro de excitacin electrnica de triptfano producido protonadaspor electrospray y se almacena en una trampa de iones de nitrgeno lquido refrigeradocuadrupolo. Mientras que elComentarios Internacional de Qumica Fsica 483Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010espectro electrnico que obtuvieron mostraron algn tipo de estructura, las caractersticas

espectrales eran rdenesde magnitud mayor que las molculas de refrigeracin en una expansin supersnica, lo quelleva a untela de juicio la eficacia de la refrigeracin en su trampa. Ms tarde se descubri que elespectro detriptfano protonada es de por s amplio debido a la rpida dinmica de estado excitado [63].Sin embargo, [62] Weinkauf los experimentos fue un importante estmulo para el estudioelectrosprayed iones en trampas fras.Este trabajo revisa el trabajo que se est realizando en el laboratorio de los autoresla combinacin de ionizacin electrospray para la produccin de biomolculas en fase gaseosaconrefrigeracin de colisin en un fro de 22 polos de trampa de iones y IR-UV resonancia dobletcnicas parasimplificar los espectros. Los objetivos principales de este trabajo ha sido comprender el valorintrnsecopropiedades de las molculas biolgicas y proporcionar pruebas de referencia fundamental dela teora, y paraesto nos fijamos en las molculas de desnudo, aislado en la fase gaseosa. Mientras que comouna prueba de la teora, laespectroscopia de biomolculas en fase gaseosa tiene una importancia completamente almargen de suanlogos de la fase condensada, los iones moleculares de capa cerrada producida porelectrospray son losmisma especie se encuentra en solucin, donde la mayora de las biomolculas tienen unacarga neta. Por otra parte, porel examen de molculas parcialmente solvatado en la fase de gas, podemos empezar aentender elpapel de disolvente en esas propiedades.2. Enfoque experimental y computacional2.1. Informacin generalLlevamos a cabo nuestros experimentos en un espectrmetro de masas de iones defabricacin casera-trampa, que se muestraesquemticamente en la figura 1. En lugar de dar una descripcin detallada de este aparato,que se puede encontrar en las publicaciones recientes [64-70], ofrecemos una brevedescripcin de laenfoque experimental seguida de una explicacin de los aspectos clave de la experimentacin


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diseo.Figura 1. [Color en lnea] Esquema del espectrmetro de masas en tndem con un refrigerado,22 polos de trampa de iones.Adaptado con permiso de [86]. Copyright 2006 Sociedad Americana de Qumica.484 T. R. Rizzo et al.

Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010Utilizamos una fuente nanospray para producir los pptidos protonadas de un acidificadosagua / metanol directamente en la atmsfera, donde se dibujan en el vacopor un vaso de metal con recubrimiento capilar. La expansin que se produce a la salida delcapilarpasa a travs de un skimmer de 1 mm de dimetro y en una trampa de iones hexapole, dondelos iones sonrecogidos para _50 ms. Una vez liberado de la hexapole, el paquete de iones resultante pasaa travs de un filtro de masas cuadrupolar para seleccionar a los de un determinado m / z. Losiones de transmisinLuego se convirti 90_ por un deflector cuadrupolo esttica, se desaceler por una serie de

lentes yguiado por un gua de iones octopolar en una trampa de iones de 22 polos, que se enfra a _6Kpor unrefrigerador de ciclo cerrado. Antes de que el paquete de iones llega a la trampa, se inyecta unpulso dehelio, dndole tiempo suficiente para equilibrarse con la temperatura de la caja trampa. Laiones colisionan con el fro de helio y la cada en el potencial efectivo y de la trampa. Nosotrosdar a los iones de aproximadamente 10 ms para enfriar y luego esperar otros 30 ms parabombearel helio antes de interrogarlos espectroscpicamente.Pasamos por un pulso de lser UV a partir de una doble frecuencia lser de colorante por el ejede la22 polos trampa de iones, la promocin de las molculas de un estado electrnico excitado deque algunosparte de ellos se disocian. A continuacin, suelte ambos el padre y los fragmentos de iones delatrampa, a su vez les 90_ en un cuadrupolo esttica segundo y pasar a travs de un anlisis decuadrupolo para seleccionar una m / z de cualquiera de un fragmento en particular o de losiones de los padres. Laiones de transmisin son detectados por un multiplicador de electrones channeltron. Unsistema electrnicoespectro se obtiene mediante el registro de la seal de iones de un fragmento en particular enfuncin de lala frecuencia del lser UV. Como se describe ms detalladamente a continuacin, se mide porinfrarrojosespectros de los iones atrapados biomolecular con IR-UV de doble resonancia.El ciclo de captura de los iones se repite en 20 Hz. Cambiamos el cuadrupolo analizarya sea para transmitir el fragmento o el padre de los ciclos alternos de captura y el uso de laseal de los padres de un disparo a normalizar la seal fragmento de la siguiente,que elimina fluctuaciones a largo plazo en la fuente nanospray.2.2. Ion generacinPoner las molculas biolgicas en la fase de gas a travs de electrospray tiene muchas ventajasparaestudios espectroscpicos. Porque se producen a temperatura ambiente y enpresin atmosfrica, esencialmente no hay fragmentacin. Parece no haber lmitecon el tamao de las molculas que se pueden producir - que van desde los aminocidos


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individualesa los virus de todo [71-73] han sido exitosamente inyecta en los espectrmetros de masas porelectrospray. Porque estamos trabajando con las biomolculas cargadas y no a los neutrales,podemos seleccionar su masa y el control de sus trayectorias, lo que nos permite orientar y / otrampa

ellos. Electrospray genera con cscara cerrada iones moleculares, ya sea protonadas odesprotonada,que es precisamente lo que se encuentra en solucin. La desventaja de estudiariones biomoleculares es la densidad del nmero relativamente bajo, lo cual es una funcintanto de laelectrospray proceso en s mismo, as como los efectos de carga espacial dentro de la trampade iones.2.3. De enfriamiento y capturaEl lineal, 22 polos de trampa de iones es el elemento clave de nuestro aparato experimental, loque permitenos para almacenar las molculas y fresco a travs de colisiones con el fro de helio. Gerlich et

al.[74-76] fue pionero en el uso de trampas de fro, de iones de radiofrecuencia y las ha aplicadoprincipalmente para estudiar laComentarios Internacional de Qumica Fsica 485Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010espectroscopia y cintica de la reaccin qumica de los iones pequeos de la importanciaastrofsica.No hay nada particularmente especial en la eleccin de 22 polos - es simplemente importantetener un nmero suficiente de ellos para que el potencial radial efectivo se fija por unparte sustancial de la trampa de dimetro [76]. Como los iones de subir la barrera de potencialradialcuando se acercan a los polos, se someten a un movimiento oscilatorio impulsado por elcampo de RF, ycolisin con helio a este punto podra calentar los iones en lugar de enfriarlos. As un potencial plano radial con las paredes ms verticales deben facilitar el enfriamiento decolisiones de iones, ya que eneste caso, el movimiento oscilatorio y por lo tanto las colisiones calentamiento, se limitar auna pequeafraccin del volumen de la trampa. Las desventajas de utilizar un mayor nmero de polos esque eliones, repartidas en un mayor volumen que resulta en una densidad de iones de baja en el ejede la trampa,y de acceso ptico a los iones atrapados en la direccin radial es limitado.2.4. Interrogatorio espectroscpicasPor lo general se inyectan 10.000 iones en la trampa de 22 polos, que tiene un volumen deactivos de _5 cm.Dada la densidad de iones de _2_103 cm_3, est claro que cualquier tipo de absorcin directaespectroscopia sera difcil si no imposible. Adems, esta densidad de iones est en el lmitede lo que puede ser detectado por fluorescencia inducida por lser (LIF), un problema que esagravada por la geometra desfavorable de la trampa de iones de 22 polos para la deteccin defotones.Necesitamos por lo tanto un medio ms sensibles de deteccin espectroscpica, y para ello seutilizanphotofragmentation.Despus de la excitacin UV de un cromforo aromtico en un pptido protonadas, algunosfraccin de los iones fragmento, ya que los rendimientos cunticos de fluorescencia son rara


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2.5. Computational methodsOur computational approach begins with a conformational search using the AMBERforce field [77] within the Macromodel package [78]. The procedure generates 50,000Dissociation thresholdInternalconversionMH+

MH A+ + B +* MH++Fragment ion signal37500 37550 37600 37650UV dissociation wavenumber/cm 1Scan UV dissociation laser(a)Fix UV dissociation laserScan IR laserDissociation thresholdInternalconversionMH+A+ MH + B +* MH++Fragment ion signal3200 3300 3400 3500 3600IR depletion wavenumber/cm

1

(b)+ +Figure 2. [Colour online] Spectroscopic schemes applied to cold biomolecular ions for measuring (a)electronic spectra of cold biomolecular ions via photofragment detection and (b) conformationspecificinfrared spectra by detecting the depletion of the UV-induced photofragment signal.International Reviews in Physical Chemistry 487Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

structures in the initial search but retains only those unique conformations under 50 kJ/ mol relative energy. For molecules with one or two amino acids, we re-optimise all of thestructures found with Macromodel using Gaussian 03 [79] at the B3LYP/6-31G** levelof theory [80,81] and carry out harmonic frequency calculations at the same level. Becausemore than one thousand conformations may be found in the initial search, we firstcalculate single-point energies of all the structures using Gaussian at the B3LYP/6-31G**level of theory, and use these energies to decide which structures merit full optimisations

and frequency calculations using B3LYP/6-31G**. In addition, all of the conformationsare classified according to the major degrees of freedom (number and type ofhydrogen bonds, aromatic ring orientation, etc.), and representative structures of eachclass are also optimised. The energies we report are zero-point corrected, and harmonicfrequencies are scaled by 0.954 for amino acids and 0.952 for helical peptides.3. Spectroscopy of protonated aromatic amino acidsThe three aromatic amino acids, tryptophan, tyrosine and phenylalanine, are naturallyoccurring candidates for probe chromophores, and it is important to understand theirspectroscopy and photophysics before using them in more complex molecules. Extensivework has been done on these aromatic amino acids in their neutral form [2,3,31,34, 82 84],but as we are investigating closed-shell ions, one must understand the effect of the chargeon the photophysics. At the same time, protonated amino acids are relatively simplesystems with which to determine the degree of cooling, and hence residual thermal

inhomogeneous broadening, in our cold ion trap. Finally, spectroscopic studies ofprotonated amino acids can be used to investigate whether IR UV double resonance canbe combined with photofragment-based detection techniques.The amino acid tryptophan has clearly been the chromophore of choice forspectroscopic studies of peptides and proteins in solution since it has a strong UVabsorption and relatively high fluorescence quantum yield. Much of the gas-phase workon neutral amino acids and peptides have also focussed on tryptophan as a chromophorein an effort to better understand its behaviour in solution in particular its nonexponentialfluorescence decay [10 12]. Despite its wide use, it is not obvious thattryptophan would be the chromophore of choice for the spectroscopic approach takenhere. The requirements for a good probe chromophore for photofragmentation-basedtechniques represent a balance between competing factors. On the one hand, one needsnon-radiative processes to occur in the excited electronic state leading to fragmentation,

allowing us to detect the absorption of radiation via the appearance of fragments. Havinga high fluorescence quantum yield means that the fraction of molecules in theelectronically excited state that fragment will be correspondingly lower and, in this


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sense, it might be an advantage to use phenylalanine, with the lowest fluorescencequantum yield, even though it has a lower oscillator strength [85]. On the other hand, if thenon-radiative processes are too fast, this will tend to broaden the electronic spectrum andinhibit our ability to resolve individual conformations. Our first goal, then, was to examinethe electronic spectra of the protonated amino acids tryptophan, tyrosine andphenylalanine. Given the extensive work done on neutral, gas-phase tryptophan [1 4],

we began our studies by examining protonated tryptophan in our cold ion trap.488 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

As in all the aromatic amino acids, protonation of the isolated molecule is expected tooccur on the amino group, forming an ammonium (NH 3 ) on the backbone. This issupported by relatively high level calculations [62] and as demonstrated below, isconsistent with the spectrum, which is little shifted from the neutral molecule. One wouldexpect protonation on the aromatic ring to cause a substantial shift of the electronic bandorigin.Figure 3 shows the photofragment excitation spectrum of TrpH in our cold ion trapobtained by collecting the fragment ions at m/z 188 (which corresponds to loss of NH 3) asa function of the wavenumber of the UV laser [86]. As a first test of our new spectroscopicapproach, these results were at first rather disappointing. While the broad, primary peak inthe spectrum is close to that of the neutral molecule, confirming that protonation does notoccur on the indole ring, its breadth suggested that the cooling in the ion trap might not becomplete. The spectrum is slightly narrower than that measured by Weinkauf in aquadrupole ion trap cooled to liquid nitrogen temperature [62] as well as that measured byTalbot et al. [87] at room temperature, but is far broader than the electronic spectrum ofthe neutral molecule measured in a supersonic expansion [3]. This initially caused us toquestion the vibrational temperature of the ions in our 22-pole ion trap and led us toperform a number of diagnostics to verify the degree of cooling. The observation that wecould form clusters of protonated amino acids with one or two helium atoms in the trap[68] suggested that the internal temperatures should be on the order of 15 K, assuming thebinding energy of helium to a protonated amino acid is similar that that of NH 4 _ He [88].A series of papers by Kang et al. [63, 89 91] helped provide insight into the reasons forthe broad TrpH spectrum. The authors used femtosecond laser pulses at 266nm to exciteprotonated tryptophan and tyrosine generated by electrospray but not cooled, and thenprobed the population of the excited state using an 800nm photon, which can excite themolecule to higher states and change the fragmentation pattern. Their results revealdrastically different excited-state lifetimes for protonated tryptophan and tyrosine.Relative fragment ion signal35,000 35,500 36,000 36,500Wavenumber/cm 1347 cm 1Figure 3. [Colour online] Electronic photofragment excitation spectrum of cold TrpH obtained bycollecting the fragment ions at m/z 188 as a function of the UV laser wavenumber. Adapted withpermission from [86]. Copyright 2006 American Chemical Society.International Reviews in Physical Chemistry 489Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

The decay of the _ _* state in TrpH exhibits a rapid drop on a timescale of 380 fsfollowed by a more complicated behaviour at later times. In contrast, protonated tyrosineexhibits a simple exponential decay of 22 ps. The observation of fast excited state decay inprotonated tryptophan suggested that the broad spectrum that we observed may not bethe result of incomplete cooling in the ion trap but could be inherently limited by the shortlifetime of the excited state. While the overall width of the main feature of TrpH (347 cm _1 ) would imply a much shorter lifetime than that measured by Kang et al. [63], itis likely that the spectrum contains contributions from a few stable conformers that mayexhibit low frequency vibrational progressions as in the case of the neutral molecule [3].If each spectral feature were broadened by 13 cm _1 , as implied by the 380 fs lifetimemeasured by Kang et al. [63], it could give rise to a broad feature with an overall width onthe order of what we observe. Moreover, one should note that the lifetimes determined byKang et al. are upper limits they cannot rule out even faster excited-state decay. Thus,our ability to form weakly bound helium clusters [68], together with the time-resolved dataof Kang et al. [63], suggests that the broad feature in the electronic spectrum of TrpH is


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not the result of incomplete cooling but results in large measure from lifetime broadening.If this were the case, then one would expect that spectral features of the other protonatedamino acids should be significantly narrower than that of TrpH .Figures 4 and 5 show electronic spectra of TyrH and PheH cooled in our 22-pole iontrap [68], both of which exhibit resolved vibronic features that reflect a significantly longerlifetime than that of protonated tryptophan, consistent with the time-resolved work of

Kang et al. [63]. These well-resolved spectra can be used as a diagnostic for the internaltemperatures of the molecules in our ion trap, demonstrated here for the case of PheH .As will be shown below, the two most prominent features in the spectrum, which occur at37,530 cm _1 and 37,541 cm _1 , correspond to the electronic band origins of two stableconformations of the molecule, and we label these band origins A 00 and B 00respectively.The inset of Figure 5 shows a blow-up of the region of the spectrum to the red of theseRelative fragment ion signal35,050 35,100 35,150 35,200 35,250 35,300 35,3502.7 cm 1A 00

B 0C 00D 00

Wavenumber (cm 1)Figure 4. [Colour online] Electronic photofragment excitation spectrum of cold TyrH recorded bydetecting the fragment at m/z 136. The labels A 00 D00 indicate the electronic band origins of fourdistinct conformers.Adapted with permission from [86]. Copyright 2006 American Chemical Society.490 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

band origins where one would expect hot-bands of low-frequency vibrational modes tooccur. One can see two features separated from A 00 and B 00 by approximately _43 cm _1 ,

which is almost identical to the spacing of the first two small peaks to the high frequencyside of the band origins. This suggests that protonated phenylalanine has a low frequencymode of _43 cm _1 in both the ground and excited electronic state. The intensity of the hotbands relative to the band origins gives us an estimate of the vibrational temperature of themolecule, although this requires us to assume a particular oscillator strength for the hotbandtransitions. If we assume that the band strength of the hot-bands, X 01, is similar tothat of its corresponding vibronic band X 10, we arrive at a vibrational temperature between11 and 16 K. This is consistent with the rotational temperature determined from a roughsimulation of the band contours. While this might not seem particularly cold, at theseinternal temperatures the populations of even the lowest frequency vibrational modes donot contribute to spectral congestion in a significant way.

With well-resolved electronic spectra such as those of Figures 4 and 5, we can useIR UV double resonance to further identify features corresponding to different stableconformations of the parent molecule by measuring an IR spectrum of each conformer.Figure 6 shows IR UV double-resonance spectra of PheH with the UV laser set on oneof the two band origins, A 00 or B 00, in Figure 5. It is evident that these IR spectra aredifferent, confirming that the two features in the electronic spectrum correspond todifferent stable conformations of the molecule. Even before measuring an IR spectrum, theelectronic spectrum of PheH suggests the presence of at least two stable conformers. Theobservation of two strong features near the band origin spaced by 10.6 cm _1 without afurther progression of peaks at roughly the same spacing suggests that these two featuresare not members of a Franck Condon progression, since by their very nature suchprogressions rarely terminate abruptly. Calculated spectra and the correspondingstructures for the two lowest-energy conformers of protonated phenylalanine are alsoRelative fragment ion signal


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37,500 37,550 37,600 37,650 37,700Wavenumber (cm 1)x10041.6 cm 143.7 cm 142.9 cm 143.6 cm 1

A 00

B0

0

Figure 5. [Colour online] Electronic photofragment excitation spectrum of cold PheH recorded bydetecting the fragment at m/z 74. The labels A 00 and B 00 indicate the electronic band origins of twodistinct conformers. To the low energy side of these band origins, a blow up of the spectrum showstwo low frequency hot bands that help determine the vibrational temperature.International Reviews in Physical Chemistry 491Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

shown in Figure 6. While the calculations do not reproduce the IR spectra exactly, theyrecover the important qualitative features two of the ammonium NH stretches arestrongly shifted to the red, one by interaction with the benzene ring and the other throughinteraction with the backbone carbonyl, while the third NH stretch is relativelyunperturbed. The calculated structures differ primarily by a rotation of the side-chainabout the C _ C _ bond.A similar treatment of TyrH reveals four rather than two conformers and acorrespondingly more complex electronic spectrum. The doubling of the number ofconformers in TyrH comes from the two possible positions of the phenol OH, whichdiffer in energy by about 120 cm _1 .Before moving on to larger peptides containing one of these chromophores, it is worthreturning to the question of protonated tryptophan. The work of Kang et al. [89] identifiedthe reason for the fast decay from the excited electronic state compared to PheH andTyrH it comes from a low crossing barrier from the _ _* state to a _ _* state that isdissociative in the NH coordinate. The difference between TrpH in the gas phase andtryptophan in solution is nevertheless curious, in that the latter has a significantfluorescence quantum yield, implying a substantially longer lifetime. One usually expectsthat excited-state lifetimes will be longer in the isolated molecule compared to solutionbecause the possibility of quenching by the solvent is removed. We investigated thisdifference by producing complexes of TrpH with water in the gas phase to observe theeffect of solvent in a stepwise manner [64]. Figure 7 shows the primary band in theelectronic spectrum of bare protonated tryptophan together with that of the singly- anddoubly-solvated species. One can clearly see a narrowing of the main band even with theaddition of a single water molecule. Upon complexation with a second water molecule, thespectral features become as narrow as those of PheH and TyrH , implying a change inlifetime of up to two orders of magnitude. As has been described in detail, this dramatic3000 3100 3200 3300 3400 3500 3600Wavenumber (cm 1)A 00

B 00

Figure 6. [Colour online] Infrared spectra of two conformers of cold of PheH obtained with theUV laser set on the band origins A 00 and B 00 in the electronic spectrum of Figure 5. Calculated spectra(B3LYP/6-31G**) are shown below the experimental spectra. The structures shown are thosederived from the corresponding calculation. Adapted with permission from [67]. Copyright 2006American Chemical Society.492 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

change comes from shifting the _ _* state relative to the _ _* state upon solvation [64].While the short excited state lifetime is caused by the charge on the ammonium, we haveshown that it does not depend upon the charge being close to the chromophore in terms ofthe number of bonds, but rather by its proximity in space. This may have importantimplications for studies using tryptophan as a spectroscopic probe in solution when the

indole ring is close to a charged group [92].From the point of view of the development of the experimental technique, theconclusions that we can draw from these studies of protonated, aromatic amino acids are


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as follows:. It is possible to cool electrosprayed ions in a 22-pole ion trap to between 11 and16 K, which is low enough that low-frequency vibrational bands are notpopulated. This essentially eliminates vibrational hot-bands from the spectra.. There is a fast decay of the indole chromophore of tryptophan in the presence of acharge. This suggests that tyrosine or phenylalanine might be better suited as

spectroscopic probes of charged, gas-phase peptides.. IR UV double-resonance can be combined with photofragment spectroscopy tomeasure conformation-specific IR spectra.We now describe the application of the same techniques to larger, more complexmolecules.34,800 35,000 35,200 35,400Wavenumber (cm 1)Wavenumber (cm 1)Wavenumber (cm 1)35,150 35,200 35,250 35,30034,800 35,000 35,200 35,400(a) TrpH +(b) TrpH +(H2O)1(c) TrpH +(H2O)2Figure 7. [Colour online] Electronic photofragment excitation spectra of TrpH with 0, 1 and 2attached water molecules. Reprinted with permission from [64]. Copyright 2006 American ChemicalSociety.International Reviews in Physical Chemistry 493Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

espectros que se obtienen son los de la conformacin de una sola molcula de inters, quefacilita en gran medida la asignacin de los espectros, as como su comparacin con lospredicciones de la teora.Hay una limitacin potencial de photofragment enfoques basados en espectroscopiacon excitacin UV. En el caso de que la conversin rpida interna despus de la excitacin UVcreauna molcula altamente vibracionalmente excitados en el estado fundamental, se espera queelunimolecular tasa de disociacin disminuye con el aumento del tamao de la molcula a lapunto que puede llegar a ser comparable a la tasa de radiacin infrarroja o colisindesactivacin con helio residual en la trampa, los cuales van a competir con la disociacin.Por lo tanto, esperamos que la deteccin espectroscpica de un solo fotn puedephotofragmentationcada vez ms difcil con el aumento de tamao molecular, aunque no est claro donde estalmite ser. En la Seccin 6, vamos a dar un ejemplo de cmo estamos superando estalimitacin.2.5. Mtodos de clculoNuestro mtodo de clculo comienza con una bsqueda de la conformacin con el AMBERcampo de fuerza [77] dentro del paquete macromodelo [78]. El procedimiento genera 50.000La disociacin del umbralInternoconversinMH +MH A + + B + * MH + +Fragmento de la seal de iones37500 37550 37600 37650UV disociacin wavenumber/cm-1Escaneo lser UV disociacin(A)Fix UV disociacin lserEscaneo lser IRLa disociacin del umbral


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InternoconversinMH +A + B + MH MH + * + +Fragmento de la seal de iones

3200 3300 3400 3500 3600IR agotamiento del nmero de onda / cm-1(B)+ +Figura 2. [Color en lnea] espectroscpicas aplicadas a planes de iones biomoleculares fro paramedir (a)espectros electrnicos de iones biomoleculares fro a travs de la deteccin y photofragmentconformationspecific (b)espectros infrarrojos mediante la deteccin de la disminucin de la seal de photofragmentinducidos por UV.

Comentarios internacional en Fsica 487 QumicaDescargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010estructuras en la bsqueda inicial, pero conserva slo las conformaciones nicas de menos de50 kJ /mol relativos de la energa. Para molculas con uno o dos aminocidos, que volver aoptimizar todos losestructuras que se encuentran con macromodelo utilizando Gauss 03 [79] en el nivel B3LYP/6-31G **de la teora [80,81] y llevar a cabo los clculos de armnicos de frecuencia en el mismo nivel.Porquems de un millar de conformaciones se puede encontrar en la bsqueda inicial, en primer lugarcalcular un solo punto las energas de todas las estructuras con Gauss en el B3LYP/6-31G **nivel de la teora, y el uso de estas energas para decidir qu estructuras mrito optimizacincompletay los clculos de frecuencia usando B3LYP/6-31G **. Adems, todas las conformacionesse clasifican de acuerdo a los grados mayores de libertad (nmero y tipo deenlaces de hidrgeno, la orientacin de anillos aromticos, etc), y las estructurasrepresentativas de cadaclase tambin estn optimizados. Las energas que se informe de punto cero corregido yarmnicafrecuencias se ajustan en 0.954 de los aminocidos y pptidos de 0.952 helicoidal.3. Espectroscopia de protonadas aminocidos aromticosLos tres aminocidos aromticos, triptfano, tirosina y fenilalanina, son, naturalmente,ocurren los candidatos a los cromforos de la sonda, y es importante para entender suespectroscopia y fotofsica antes de usarlos en molculas ms complejas. Extensotrabajo se ha hecho en estos aminocidos aromticos en su forma neutral [2,3,31,34, 82-84],pero como estamos investigando con cscara cerrada iones, uno debe entender el efecto de lacargaen el fotofsica. Al mismo tiempo, protonadas aminocidos son relativamente simplessistemas que permitan determinar el grado de enfriamiento, y por lo tanto trmica residualhomognea posible ampliacin, en nuestra trampa de iones fro. Finalmente, los estudiosespectroscpicos deprotonadas aminocidos se pueden utilizar para investigar si IR-UV de doble resonancia puedecombinarse con tcnicas de deteccin basadas en photofragment.El aminocido triptfano ha sido claramente el cromforo de eleccin paraLos estudios espectroscpicos de pptidos y protenas en solucin, ya que tiene un fuerte UV


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absorcin y rendimiento de fluorescencia relativamente alto cuntica. Gran parte del trabajoen fase gaseosaneutral en aminocidos y pptidos tambin se centr en triptfano como un cromforoen un esfuerzo por entender mejor su comportamiento en la solucin - en particular, sunonexponential

decaimiento de fluorescencia [10-12]. A pesar de su amplio uso, no es obvio quetriptfano sera el cromforo de eleccin para el enfoque espectroscpicas tomadasaqu. Los requisitos para un cromforo sonda bueno para photofragmentation basado entcnicas representan un equilibrio entre los factores de la competencia. Por un lado, senecesitano radiativa los procesos que se produzcan en el estado electrnico excitado que conduce a lafragmentacin,lo que nos permite detectar la absorcin de la radiacin a travs de la aparicin defragmentos. Despus de haberun alto rendimiento cuntico de fluorescencia significa que la fraccin de molculas en elestado electrnicamente excitado ese fragmento ser proporcionalmente inferior y, en este

sentido, podra ser una ventaja para el uso de fenilalanina, con el menor fluorescenciarendimiento cuntico, a pesar de que tiene una fuerza de oscilador de baja [85]. Por otro lado,si elno radiativa procesos son demasiado rpidos, lo que tender a ampliar el espectro electrnicoyinhiben nuestra capacidad para resolver conformaciones individuales. Nuestro primer objetivoera, pues, a examinarlos espectros electrnicos de los aminocidos protonadas triptfano, tirosina yfenilalanina. Dado el amplio trabajo realizado en el neutro, en fase gaseosa triptfano [1-4],comenzamos nuestros estudios mediante el examen de triptfano protonadas en la trampa deiones fro.488 T. R. Rizzo et al.Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010Como en todos los aminocidos aromticos, la protonacin de la molcula aislada se esperaqueocurren en el grupo amino, formando una de amonio (NH3) en la columna vertebral. Escon el apoyo de los clculos de nivel relativamente alto [62] y como se demuestra acontinuacin, seconsistente con el espectro, que es un poco pasado de la molcula neutra. UnoEsperamos protonacin en el anillo aromtico para causar un cambio sustancial de la bandaelectrnicaorigen.La Figura 3 muestra el espectro de excitacin photofragment de TrpH en nuestra trampa deiones froobtenida por la recogida de los fragmentos de iones de m / z 188 (que corresponde a laprdida de NH3), comoen funcin del nmero de onda del lser UV [86]. Como primera prueba de nuestro nuevoespectroscpicasenfoque, los resultados fueron al principio ms bien decepcionante. Mientras que el picoancho, en primariael espectro es similar a la de la molcula neutra, lo que confirma que la protonacin nose producen en el anillo de indol, su amplitud sugiri que el enfriamiento en la trampa deiones podra no sercompleta. El espectro es ligeramente ms estrecho que el medido por Weinkauf en uncuadrupolo de trampa de iones se enfra a temperatura de nitrgeno lquido [62], as como la


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medida porTalbot et al. [87] a temperatura ambiente, pero es mucho ms amplio que el espectroelectrnico dela molcula neutra se mide en una expansin supersnica [3]. Este principio nos hizocuestin de la temperatura de vibracin de los iones en la trampa de iones de 22 polos y nos

llev arealizar una serie de pruebas para verificar el grado de enfriamiento. La observacin de quenospodran formar grupos de protonadas aminocidos con uno o dos tomos de helio en latrampa[68] sugiere que la temperatura interna debe ser del orden de 15 K, suponiendo que elenerga de enlace de helio a un cido amino protonado es similar que la de NH4 _ El [88].Una serie de documentos por Kang et al. [63, 89-91] ayud a dar una idea de las razones porlasel espectro TrpH amplio. Los autores usaron pulsos de lser de femtosegundo en 266 nm

para excitartriptfano y la tirosina protonadas generada por electrospray, pero no se enfra, y luegoprobaron la poblacin del estado excitado con un fotn de 800 nm, lo que puede excitar a losmolcula a estados superiores y cambiar el patrn de fragmentacin. Sus resultados revelandrsticamente diferente en estado excitado vida para el triptfano y la tirosina protonada.Fragmento de seal relativa de iones35.000 35.500 36.000 36.500Wavenumber/cm-1347 cm-1Figura 3. [En lnea Color] espectro electrnico de excitacin photofragment de fro TrpHobtenidos porrecoger los fragmentos de iones de m / z 188 en funcin del nmero de onda lser UV.Adaptado conpermiso de [86]. Copyright 2006 Sociedad Americana de Qumica.Comentarios Internacional de Qumica Fsica 489Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010La decadencia de la _-_ * Estado en TrpH exhibe una rpida cada en un plazo de 380 fsseguido por un comportamiento ms complicado en los ltimos tiempos. Por el contrario, latirosina protonadasmuestra un decaimiento exponencial simple de 22 ps. La observacin de la descomposicinrpida en estado excitadotriptfano protonadas sugiri que el amplio espectro que hemos observado no puede serel resultado del enfriamiento incompleta en la trampa de iones, pero podra serinherentemente limitada por el cortotiempo de vida del estado excitado. Mientras que el ancho total de la caracterstica principalde TrpH(347 cm_1) implicara una vida til mucho ms corta que la medida por Kang et al. [63], queEs probable que el espectro contiene contribuciones de algunos conformistas estable quepuedepresentan las progresiones de baja frecuencia vibratoria como en el caso de la molcula neutra[3].Si cada caracterstica espectral se ampliaron en un 13 cm_1, como se deduce de la vida fs 380medida por Kang et al. [63], podra dar lugar a una caracterstica general con una anchura totaldeel orden de lo que observamos. Por otra parte, cabe sealar que el tiempo de vidadeterminado por


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Kang et al. son los lmites superiores - que no se puede descartar an ms rpido en estadoexcitado decadencia. Por lo tanto,nuestra capacidad para formar grupos dbilmente helio [68], junto con los datos de tiemporesueltode Kang et al. [63], sugiere que la funcin amplia en el espectro electrnico de TrpH es

no el resultado del enfriamiento incompleto, pero la ampliacin de los resultados en granmedida de toda la vida.Si este fuera el caso, entonces uno podra esperar que las caractersticas espectrales de losotros protonadasaminocidos debe ser significativamente menor que el de TrpH.Las figuras 4 y 5 muestran los espectros electrnicos de TyrH y PheH enfra en nuestros 22polos de ionestrampa [68], los cuales presentan caractersticas resuelto vibronic que reflejan unasignificativamente mayorvida que la de triptfano protonadas, de conformidad con la resolucin temporal de trabajoKang et al. [63]. Estos espectros bien resueltos se puede utilizar como un diagnstico para el

mercado interiortemperaturas de las molculas en nuestra trampa de iones, ha demostrado aqu en el caso dePheH.Como se ver ms adelante, las dos caractersticas ms importantes en el espectro, que seproducen en37.530 y 37.541 cm_1 cm_1, corresponden a los orgenes de la banda electrnica de dosestablesconformaciones de la molcula, y la etiqueta de estos orgenes banda A00 y B00, respectivamente.El recuadro de la Figura 5 se muestra un blow-up de la regin del espectro hacia el rojo deestosFragmento de seal relativa de iones35.050 35.100 35.150 35.200 35.250 35.300 35.3502,7 cm-1A 00B 0C 00D 00Nmero de onda (cm-1)Figura 4. [En lnea Color] espectro electrnico de excitacin photofragment de fro TyrHregistrados pordetectar el fragmento de m / z 136. Las etiquetas A00-D00 indican los orgenes de la banda electrnica de cuatrodistintas conformers.Adapted con permiso de [86]. Copyright 2006 Sociedad Americana deQumica.490 T. R. Rizzo et al.Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010orgenes la banda donde se podra esperar en caliente bandas de baja frecuencia para losmodos de vibracinocurrir. Uno puede ver dos caractersticas separadas de A00 y B0


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0 en aproximadamente _43 cm_1,que es casi idntica a la separacin de los dos primeros picos pequeos a la alta frecuencialado de los orgenes de la banda. Esto sugiere que la fenilalanina protonadas tiene unafrecuencia bajamodo de _43 cm_1 tanto en el suelo y el estado electrnico excitado. La intensidad del calor

bandas en relacin con los orgenes de banda nos da una estimacin de la temperatura devibracin de lamolcula, aunque esto nos obliga a asumir una fuerza particular para el oscilador hotbandtransiciones. Si asumimos que la fuerza de la banda de las bandas en caliente, X01, es similar a lade su correspondiente banda X1 vibronic0, se llega a una temperatura de vibracin entre11 y 16 K. Esto es consistente con la temperatura de rotacin determina a partir de un durosimulacin de los contornos de la banda. Aunque esto puede no parecer particularmente fro,en estosla temperatura interna de las poblaciones de incluso los modos de menor frecuencia de

vibracin seno contribuyen a la congestin del espectro de una manera significativa.Con los espectros bien resueltos electrnicos, tales como las de las figuras 4 y 5, se puedeutilizarIR-UV de doble resonancia para determinar con ms precisin las caractersticascorrespondientes a las distintas estableconformaciones de la molcula mediante la medicin de un espectro IR de cada conformacin.La figura 6 muestra IR-UV de doble resonancia espectros de PheH con el lser UV encuentraen unade los dos orgenes de la banda, A00 o B00, En la Figura 5. Es evidente que estos espectros IR sediferentes, lo que confirma que las dos caractersticas en el espectro electrnico correspondenadiferentes conformaciones estables de la molcula. Incluso antes de la medicin de unespectro de IR, elespectro electrnico de PheH sugiere la presencia de al menos dos confrmeros estables. Laobservacin de dos caractersticas fuertes cerca de la banda de origen separados por 10,6cm_1 sinprogresin de los picos en aproximadamente la misma distancia sugiere que estas doscaractersticasque no son miembros de una progresin de Franck-Condon, ya que por su naturaleza, muyprogresiones rara vez terminado abruptamente. Espectros calculados y la correspondienteestructuras de los dos confrmeros de menor energa de la fenilalanina protonadas tambinFragmento de seal relativa de iones37.500 37.550 37.600 37.650 37.700Nmero de onda (cm-1)x10041,6 cm-143,7 cm-142,9 cm-143,6 cm-1A 00B00


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Figura 5. [En lnea Color] espectro electrnico de excitacin photofragment de fro PheHregistrados pordetectar el fragmento de m / z 74. Las etiquetas A00 y B00 indican los orgenes de la banda electrnica de dos

confrmeros distintos. Para el lado de baja energa de estos orgenes la banda, un golpe arribadel espectro muestrados bandas de frecuencia baja caliente que ayudan a determinar la temperatura de vibracin.Comentarios Internacional de Qumica Fsica 491Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010muestra en la Figura 6. Mientras que los clculos no se reproducen los espectros IRexactamente,recuperar las caractersticas cualitativas importantes - dos de los tramos de amonio NHfuertemente desplazado hacia el rojo, uno por la interaccin con el anillo de benceno y la otraa travs deinteraccin con el carbonilo columna vertebral, mientras que el tercer tramo de NH es

relativamenteimperturbable. Las estructuras calculadas difieren principalmente por una rotacin de lacadena lateralsobre el vnculo C_-C_.Un tratamiento similar de TyrH revela cuatro en lugar de dos confrmeros y unespectro electrnico correspondientemente ms complejas. La duplicacin del nmero deconfrmeros en TyrH proviene de las dos posiciones posibles de la OH fenol, quedifieren en la energa de unos 120 cm_1.Antes de pasar a los grandes pptidos que contienen uno de estos cromforos, vale la penaVolviendo a la pregunta de triptfano protonada. La obra de Kang et al. [89] identificla razn de la descomposicin rpida del estado electrnico excitado en comparacin conPheH yTyrH - se trata de una barrera de baja cruce de la _-_ * Estado a una _-_ * estado que sedisociativos en la coordinacin de NH. La diferencia entre TrpH en la fase gaseosa ytriptfano en la solucin es, sin embargo curioso, en que este ltimo tiene un impactosignificativorendimiento cuntico de fluorescencia, lo que implica una vida mucho ms tiempo. Por logeneral se espera queque en estado excitado vida ser ms larga de la molcula aislada en comparacin consolucinporque la posibilidad de extincin por el disolvente se ha eliminado. Hemos investigado estadiferencia mediante la produccin de los complejos de TrpH con agua en la fase gaseosa paraobservar elefecto del disolvente en una forma gradual [64]. La figura 7 muestra la banda principal en elespectro electrnico de triptfano protonadas desnudo junto a la de los mono-ydoblemente solvatados especies. Uno puede ver claramente un estrechamiento de la bandaprincipal, incluso con laAdems de una sola molcula de agua. Tras la formacin de complejos con una molcula deagua en segundo lugar, lacaractersticas espectrales a ser tan estrechas como las de PheH y TyrH, lo que implica uncambio en lavida til de hasta dos rdenes de magnitud. Como se ha descrito en detalle, esta espectacular3000 3100 3200 3300 3400 3500 3600Nmero de onda (cm-1)A 00


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B 00Figura 6. [Color en lnea] infrarrojos espectros de dos confrmeros de fro de PheH obtenidoscon elLser UV conjunto sobre los orgenes de banda A0

0 y B00 en el espectro electrnico de la figura 5. Espectros calculados(B3LYP/6-31G **) A continuacin se muestran los espectros experimentales. Las estructurasse muestran son lasderivados de los clculos correspondientes. Adaptado con permiso de [67]. Copyright 2006American Chemical Society.492 T. R. Rizzo et al.Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010el cambio viene de cambiar el _-_ * Estado en relacin con el _-_ * en estado de solvatacin[64].Mientras que el corto tiempo de vida del estado excitado es causada por la carga de amonio,

hemosdemostrado que no depende de la carga de estar cerca de los cromforos en trminos deel nmero de enlaces, sino ms bien por su proximidad en el espacio. Esto puede tenerimportantesimplicaciones para los estudios de uso de triptfano como sonda espectroscpica en unasolucin cuando elanillo de indol est cerca de un grupo encargado de [92].Desde el punto de vista del desarrollo de la tcnica experimental, laconclusiones que podemos extraer de estos estudios de los cidos protonados, aminocidosaromticos sonde la siguiente manera:. Es posible enfriar los iones electrosprayed en una trampa de iones de 22 polos de entre 11 y16 K, que es suficientemente bajo para que las bandas de baja frecuencia de vibracin no sonpobladas. Esto esencialmente elimina la vibracin en caliente las bandas de los espectros.. Hay un decaimiento rpido del cromforo indol de triptfano en la presencia de unde carga. Esto sugiere que la tirosina o fenilalanina podra ser ms adecuada comosondas espectroscpicas de carga, en fase gaseosa pptidos.. IR-UV de doble resonancia puede ser combinada con la espectrometra de photofragmentmedida de la conformacin especfica de los espectros IR.Ahora describir la aplicacin de las mismas tcnicas para grandes, ms complejosmolculas.34.800 35.000 35.200 35.400Nmero de onda (cm-1)Nmero de onda (cm-1)Nmero de onda (cm-1)35.150 35.200 35.250 35.30034.800 35.000 35.200 35.400(A) TrpH +(B) TrpH + (H2O) 1(C) TrpH + (H2O) 2Figura 7. [Color en lnea] electrnica photofragment espectros de excitacin de TrpH con 0, 1y 2las molculas de agua adjunto. Reproducido con permiso del [64]. Copyright 2006 AmericanChemicalLa sociedad.


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Comentarios Internacional de Qumica Fsica 493Descargado por: [Universidad Pablo de Olavide] En: 08:33 09 de enero 2010 4. The scaling of spectral complexity with increasing molecular sizeThe electronic spectra of the protonated amino acids TyrH and PheH , while complexcompared to those of small molecules typically studied by gas-phase spectroscopy, are stillrather simple in that the region near the vibronic band origin is well-resolved, allowing theselection of individual conformers for measuring conformer-specific IR spectra. If oneapplies these techniques to molecules of increasing size, the question arises as to whetherthe electronic spectra will be too complex to resolve individual features.Consider the factors that could potentially contribute to the complexity of theelectronic spectrum of a large molecule:(1) thermal inhomogeneous broadening;(2) spectral broadening from short excited state lifetimes;(3) a large number of Franck Condon active low-frequency vibrations;(4) conformational heterogeneity.We have demonstrated that for protonated amino acids we can eliminate thermalinhomogeneous broadening through collisions with cold helium in an ion trap and that thecooling process is complete within the first 5 7 ms, after which the spectra no longerchange [68]. Given that a significant number of cooling collisions continue to occur overthe first 30 40 ms, it seems that the trap still has considerable unused cooling capacity.Moreover, while larger molecules contain more internal energy, the collision cross section(and hence the collision rate) with cold helium in the trap also increases, as does thenumber of low frequency modes through which it is easy to lose vibrational energy. Thusfor fixed helium density the ultimate temperature achieved should only increase slowlywith increasing molecular size.The second factor is related the specific dynamics of the probe chromophore. We haveshown that the indole chromophore of tryptophan exhibits fast excited-state decay andbroadened spectra when in proximity to a charged group. While this will remain aproblem, we can simply choose to use other chromophores, such as tyrosine orphenylalanine, when this factor becomes limiting.The third factor may be an important one. The number of vibrational modes increasesrapidly with the number of amino acid residues in a peptide. The question that we need toaddress is how many of these modes will have strong Franck Condon activity, particularlythose of low frequency that could give rise to vibrational progressions near the band originthat will prevent the resolution of individual conformers. There is some reason to believethat not all modes will appear in the spectrum. The vibrational modes that have strongFranck Condon factors will be those for which the bond lengths and angles associatedwith the vibrational motion change in geometry upon excitation to the excited electronicstate. Because we are using the aromatic side chains of amino acids as chromophores, oneexpects that most of the change in electron density in the excited state will be centredaround these chromophores, and thus those bond lengths and angles near to or associatedwith the aromatic moiety are the ones most likely to change. Thus vibrational modesinvolving coordinates remote from the chromophore are unlikely to feel the change in electronic excitation and hence may not appear as a vibrational progression in theelectronic spectrum.The last factor that one has to consider is conformational heterogeneity that is,the fact that as molecules become larger the number of possible stable494 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

conformations increases. As we cool molecules and freeze out different conformations inour ion trap, the electronic spectrum of the molecule may be extremely congested, since theprecise frequency of the vibronic band origin depends upon the conformation. There islittle we can argue on this point in terms of a priori expectations we are simply left to trythe experiment and observe the degree of complexity. Ultimately we can reduce thenumber of stable conformations in our ion trap by pre-selecting the ions according to theirmobility in a helium buffer gas before we trap them. This work is currently being pursuedin our laboratory.To test the degree of spectral complexity in a series of molecules of increasing size, we

chose peptides containing a single tyrosine chromophore and increasing numbers ofalanine residues. The electronic spectra of this series of molecules are shown in Figure 8.


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As one can see from this progression, although the number of vibrational modes of themolecules increases by more than a factor of three, the electronic spectrum does notbecome significantly more complex, and the vibrational band origin seems to remain wellresolved. We can draw several conclusions from this simple series of spectra, although onemust be careful about generalising them. First, while there is increasing complexity withincreasing size, it seems in this case not to be monotonic, and the number of Franck

Condon active vibrational modes does not seem to track the total number of vibrationalmodes directly. Second, the fact that the band origin remains well resolved seems to reflectthe lack of extremely low frequency modes that couple to the electronic transition. Whilesuch modes certainly exist, they would have to involve the overall motion of the peptidebackbone in such a way as to change the interaction with the chromophore uponvibration. The relative sparsity of the spectrum in the first 20 cm _1 after the electronicband origin may simply mean that the lowest frequency modes do not couple to thechromophore.Finally, the simplicity of the spectrum near the band origin also implies that there arenot large numbers of different stable conformers present in the ion trap. While infraredspectra of TyrH revealed that four conformers contributed to the electronic spectrum[67], the electronic spectrum of H AlaTyr appears simpler, and we believe that this is dueto a reduced number of stable conformations [66]. Because the molecule is more flexible,

the protonated ammonium group can more easily be in contact with the aromatic ring ofthe tyrosine and maximise this stabilising interaction. A conformer-selected infraredspectrum of this dipeptide is shown in Figure 9, together with a calculated spectrum andthe conformation giving rise to it. The close proximity of the ammonium group to thearomatic ring, which is not possible in the bare amino acid, verifies the importance ofthe cation _ interaction in stabilising certain conformations.Even for the larger members of this series, the band origin does not seem to be overlycongested by the presence of a large number of conformers. Whether this behaviour can begeneralised to molecules of even larger size remains to be seen, but the results presentedabove suggests that our spectroscopic techniques can be applied to molecules at least thesize of pentapeptides.5. Spectroscopy of helical peptidesHaving demonstrated that our experimental approach can generate well-resolved

electronic and vibrational spectra of small peptides, we now show the results ofInternational Reviews in Physical Chemistry 495Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

applying the same techniques to a series of molecules that we expect to be helical in thegas phase [65,69,93]. The goal of this study was to identify spectroscopic signatures ofhelices and to test the ability of theory to predict them.The basis of this work comes from the ion mobility studies of Jarrold et al. [94,95], whoexamined a series of alanine-containing peptides, Ac-(Ala) n-LysH , Ac-LysH -(Ala) n,35,050 35,100 35,150 35,200 35,250Wavenumber (cm 1)34,500 34,550 34,600 34,650 34,700Wavenumber (cm 1)35,850 35,900 35,950 36,000 36,050Wavenumber (cm 1)35,050 35,100 35,150 35,200 35,250Wavenumber (cm 1)

(a) TyrH+(b) H+AlaTyr(c) H+AlaAlaTyr(d) H+AlaAlaTyrAlaAlaFigure 8. [Colour online] Electronic photofragment excitation spectra of a series of tyrosinecontainingpeptides; (a) TyrH ; (b) H AlaTyr; (c) H AlaAlaTyr; (d) H AlaAlaTyrAlaAla.496 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

and (Ala) nH, as a function of the number of alanine residues, n. By measuring the relativedrift times through helium, they determine the average cross section of each of thesespecies, and in comparison with molecular dynamics calculations they can detect thedifference between helical and non-helical structures. With the N-terminus protected theyfind that helix formation occurs only when the lysine is at the C-terminus. Putting it at theN-terminus or eliminating it altogether gives rise to non-helical structures. The protonated

side-chain of the lysine plays two roles: it is involved in hydrogen bonding with threecarbonyl groups on the backbone in such a way as to induce helix formation, and itstabilises the helical structure through a favourable electrostatic interaction with the helix


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macro-dipole.For us to measure conformer-specific IR spectra of these alanine-based peptides weneed to introduce a UV chromophore in a way that does not interfere with the formationof secondary structure, and we do this by substituting a phenylalanine for the N-terminalalanine reside. Figure 10 shows the electronic spectrum of two such peptides with lysine atthe C-terminus: Ac-Phe-(Ala) 5-LysH and Ac-Phe-(Ala) 10-LysH , which according to

the ion mobility studies should form a helix [95], and one with lysine at the N-terminus,Ac-LysH -Phe-(Ala) 10 , which should not form a helix.The first thing to notice is the relative simplicity of the UV spectra in all cases. Despitethe fact that these are larger than the tyrosine-containing peptides shown in Figure 8, theelectronic spectra seem, if anything, even simpler. It is clear that at most a small fraction ofthe low-frequency vibrational modes carry strong Franck Condon activity.Figure 11 shows conformer-specific infrared spectra obtained by setting the UV laser ata particular band in the electronic spectrum and detecting the dips while scanning the IROPO. The spectra of Figure 11a and b result from monitoring the two major features,3000 3200 3400 36000102030

Wavenumber (cm 1)Percent depletionPhenolOHamideNHFree ammoniumNHBound ammoniumNHCaboxyllicacid OH

Figure 9. [Colour online] Conformer-specific IR spectrum of H AlaTyr recorded with the UV laserset to the electronic band origin at 34525.2 cm _1 together with the calculated spectrum (B3LYP/ 6-31G**) and corresponding molecular structure for the lowest energy conformer.International Reviews in Physical Chemistry 497Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010

marked A and B in Figure 10a, for Ac-Phe-(Ala) 5-LysH ; Figure 11c is recorded with theUV laser frequency fixed on peak A in Figure 10b for Ac-Phe-(Ala) 10-LysH , andFigure 11d is obtained with the UV frequency set on the major peak in the spectrum ofAc-LysH -Phe-(Ala) 10 shown in Figure 10c. There are several important features to noticein the infrared spectra of Figure 11a and b. First, the simple fact that these spectra aredifferent indicates that the features in the electronic spectra used to obtain themcorrespond to different stable conformers. One can thus distinguish conformers relativelyeasily using such IR spectra, even in molecules of this size.Fragment depletion3200 3300 3400 3500 3600Wavenumber (cm 1)(b)(a)(c)(d)

Figure 11. [Colour online] Infrared spectra of (a) conformer A of Ac-Phe-(Ala) 5-Lys-H ,(b) conformer B of Ac-Phe-(Ala) 5-Lys-H , (c) conformer A of Ac-Phe-(Ala) 10 -Lys-H and(d) Ac-LysH -Phe-(Ala) 10 .Relative fragmentation yield37,500 37,550 37,600Wavenumber (cm 1)(a)(b)(c)A BAC DBFigure 10. [Colour online] Electronic photofragment excitation spectra of (a) Ac-Phe-(Ala) 5-Lys-H, (b) Ac-Phe-(Ala) 10 -Lys-H , and (c) Ac-LysH -Phe-(Ala) 10 . The labels in (a) and (b) denote

band origins of different conformers.498 T. R. Rizzo et al.Downloaded By: [Universidad Pablo de Olavide] At: 08:33 9 January 2010


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The peak near 3575 cm _1 can be assigned to the free carboxylic OH stretch of thelysine at the C-terminus. The presence of a free OH stretch band is already evidence thatthe molecule may be helical, since if it were to adopt a globular structure, it is likelythat the carboxylic OH would find itself in a hydrogen-bonding interaction that wouldshift the corresponding band to lower wavenumber by an amount depending upon thestrength of the interaction.

The bands in the region 3300 3500 cm _1 arise from amide NH stretch vibrations. Theappearance of seven bands in this region is consistent with fact that there are seven amidestretches and indicates that we are able to see all of them. These bands vary in both theirposition and their width those shifted further to the red seem to be broader. Thenarrowest bands are only 2 cm _1 in width (before deconvolution of the OPO linewidth,which is _1cm _1 ), while the wider amide bands approach 6 cm _1 . If the molecule is indeedhelical, the narrow, higher frequency amide NH stretch bands are likely to be those thatreside at the ends of the molecule and which do not participate in hydrogen bonding alongthe helix axis, whereas the red-shifted bands should come from the NH residues in themiddle of the helix. This is confirmed by the observation that adding five additionalalanines results in additional features in this red-shifted region, as shown in the IRspectrum of Ac-Phe-(Ala) 10 -LysH (Figure 11c). Notice also that this larger molecule stillexh

Traduccion Articulos VCD Thomas R. Rizzo

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